A Single Point Mutation within the Coding Sequence of Cholera Toxin B Subunit Will Increase Its Expression Yield

Background: Cholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector. Methods: ctxB fragment from Vibrio cholerae O1 ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. coli Top 10F' and cultured on LB agar plate containing ampicillin. Sequence analysis confirmed the mature ctxB gene sequence and the mutant one in both constructs which were further subcloned to pQE-30 vector. Both constructs were subsequently transformed to E. coli M15 (pREP4) for expression of mature and mutant rCTB. Results: SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 hours after induction and Western-blot analysis confirmed the presence of rCTB in blotting membranes. The expression of mutant rCTB was much higher than mature rCTB, which may be the result of serine-to-threonine substitution at position 128 of mature rCTB amino acid sequence created by PCR mutagenesis. The mutant rCTB retained pentameric stability and its ability to bind to anti- cholera toxin IgG antibodies. Conclusion: Point mutation in ctxB sequence resulted in over-expression of rCTB, probably due to the increase of solubility of produced rCTB. Consequently, this expression system can be used to produce rCTB in high yield.


INTRODUCTION
holera is a severe diarrheal disease which still remains as an epidemic or endemic threat in many parts of the world, particularly in developing countries [1]. Vibrio cholerae is classified into more than 200 serotypes on the basis of somatic antigens [2]. Seven cholera epidemics which are caused by only O1 and O139 serogroups have occurred, and its outbreaks continue to occur in Iran and other developing countries [3][4][5]. Cholera is mainly caused in countries with poor sanitations, and many attempts have been performed to develop an improved vaccine [6].
Cholera symptoms are mainly caused by cholera toxin [7], an 85-kDa protein. This protein is composed of two subunits: a single A subunit (cholera toxin A [CTA]) which is responsible for activation of adenylate cyclase in the intestinal cells, and B subunit (cholera toxin B [CTB], 11.6 kDa), which binds the respective holotoxin to its intestinal receptor (ganglioside GM1 [monosialotetrahexosylganglioside] [8,9]. CTB has recently attracted many interests as an adjuvant for various other peptide or carbohydrate antigens [10,11]. It comprises a transmucosal carrier delivery system for induction of oral tolerance when conjugated to antigens and allergens [7,12]. When CTB is chemically or genetically conjugated to poor immunogenes, it can elicit serum and secretory antibodies against the fused antigens [13,14]. Vaccination against cholera is a powerful prevention strategy, because it can provide long-term protective immunity [15][16][17]. Based on this realization, a variety of vaccines against cholera were developed that divided into two principal kinds, the killed and live attenuated Vibrio cholerae vaccines [6]. Various hosts have been used to develop a high-level expression system for producing recombinant CTB (rCTB), but most of these attempts were failed. The aim of this study was to evaluate two different strategies for C

Expression of recombinant cholera toxin B subunit.
Transformed E. coli M15 (pREP4) cells (Qiagen, Canada) harboring pQE-ctxB construct were grown in LB medium containing ampicillin (100 µg l -1 ) and incubated at 37°C with shaking (200 rpm) until optimal density at 600 nm reached to 0.6-0.8. The IPTG (Sigma-Aldrich, Germany) was added to the culture at a final concentration of 1 mM. Additional 5-h incubation with shaking was carried out, and culture sampling was performed in each hour. Bacterial cells were harvested by centrifugation at 4000 ×g at 4°C for 20 min.

SDS-PAGE analysis and Western-blotting.
The pellet of bacterial cells was lysed by 200 µl lysis buffer (25 mM TRIS-Cl and 2 mM EDTA, pH 7.6) for each sample following sonication. Lysed pellets were mixed with sample buffer, boiled for 10 min and electrophoresed on two separate SDS-PAGE gels (15% wv -1 ) under the same running conditions. One gel was stained with Coomassie Brilliant Blue R-250 (1% wv -1 ), and the other one was subjected to blotting onto poly (vinylidene difluoride) membrane (Hi-bond Amersham Biosciences, Piscataway, NJ, USA). Western-blot analysis was performed using 1:1000 dilution of rabbit polyclonal anti-cholera toxin antibody (Sigma-Aldrich, Germany) in PBS and 1:10000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, Germany) in PBS as primary and secondary antibodies, respectively. The production of rCTB was detected with bound antibodies using a chemiluminescent substrate, electrophoresis chemiluminescent (Hi-bond Amersham Biosciences, Piscataway, NJ, USA) and then exposed to Kodak X-OMAT Blue Autoradiography Film. The amount of expressed rCTB was evaluated using FireReader D56 software (UVI Tec, UK).

Over-expression of mutant recombinant cholera toxin B subunit. Mutant rCTB gene was design via
replacing the 383 th nucleotide of mature ctxB gene (guanine instead of cytosine). PCR reaction was carried out using ctxB-F (5'GCG TCA TGA TTA AAT TAA  AAT TTG GTG TTT TTT TTA CAG TTT  TAC3')/ctxB-R (5'CGC CTC GAG GGA ACC GCG TGG CAC CAG ATT TGC CAT AGT AATTG 3') primers. The base in bold in reverse primer sequence is the substitution base. Mutant ctxB fragment was cloned in pGEM and subcloned in pQE-30 for expression the mutant rCTB as described previously. Expression of this new mutant rCTB was analyzed with SDS-PAGE gel (15% wv -1 ) and confirmed by Western-blot analysis. The amount of produced mutant rCTB was evaluated using FireReader D56 software (UVI Tec, UK).

Amplification and cloning of ctxB.
The expected PCR product, a single band of 390 bp for ctxB gene, was obtained (Fig. 1A). The recombinant pGEM-T-ctxB plasmid extracted from E. coli Top10 was digested with EcoRI. Two bands of 3,000 bp and 390 bp were obtained related to pGEM-T vector and ctxB gene, respectively (Fig. 1B). The pGEM-T-ctxB was double digested with BspHI and XhoI restriction enzymes; approximately a 390-bp band corresponding to ctxB was appeared in agarose gel (Fig. 1C).

Construction of mutant ctxB sequence.
Mutant ctxB gene was produced using designed primers with a point mutation in reverse primer sequence, which belonged to 383 th nucleotide of mature ctxB gene (cytosine was replaced with guanine in mutant ctxB gene). This point mutation finally produced threonine amino acid instead of serine in mutant rCTB protein. Both of these amino acids had polar, uncharged R group in their chemical structure (CH 2 OH for serine and CHOHCH 3 for threonine). The sequence of mutant ctxB was confirmed by DNA sequence analysis.

Expression of mature and mutant recombinant cholera toxin B subunit.
Two constructs of pQE vector, one containing native ctxB fragment and the other containing mutant ctxB gene were transformed separately into E. coli M15 (pREP4). Time gradient was applied to recognize the best time for producing maximum amount of rCTB in both constructs. The best condition to produce rCTB in both constructs was determined at 37°C, 1 mM concentration of IPTG and 5 hours after induction ( Fig. 2A and 2B). A major band of approximately 14.5 kDa corresponding to rCTB was   observed on SDS-PAGE analysis of both constructs. Comparison of SDS-PAGE analysis of the two different constructs using FireReader D56 software (UVI Tec, UK) demonstrated that the expression of mutant rCTB was assessed to be approximately 80 mg l -1 , while the expression of mature rCTB was about 7.8 mg l -1 (approximately 10 fold higher than mature rCTB in the same conditions) ( Fig. 2A and 2B). Overexpression of mutant rCTB may be occurred due to replacement of serine for threonine in CTB protein structure via point mutation. Antigenicity of expressed proteins was confirmed by immunoblotting using anticholera toxin IgG. The over-expression of mutant rCTB in contrast with mature rCTB was observed after exposure protein bands on a blue autoradiography film ( Fig. 3A and 3B).

DISCUSSION
CTB has been extensively studied as an immunogen or adjuvant in intestinal and nasal mucosal sites [15,18]. It has ability to deliver covalently attached antigens to the mucosal cells via binding to GM 1 ganglioside receptor on the surface of epithelial cells [19,20]. CTB was used as an adjuvant to increase the immune response level in many different vaccines specially vaccines against toxigenic Vibrio cholerae strains [21][22][23]. Many efforts have been performed to produce CTB in E. coli [24], Lactobacillus and Bacillus brevis [25,26] or even Vibrio cholerae strains lacking the CTA gene [7]. Yeasts (Saccharomyces cerevisiae) and plants (such as tomato, potato, and tobacco) have been also used to produce rCTB, but the amount of expressed protein is too low in former systems [27]. The sequence analysis of cholera toxin revealed a number of mutations at different positions on the genome sequence of both cholera toxin subunits across different serogroups. Some of these mutations are non-synonymous such as polar to non-polar or vice-versa. However, some other, especially at structurally related positions, may be important for understanding the pathogenesis of different serogroups or for improvement of recombinant cholera toxin proteins to produce vaccines against multiple serogroups [28]. The substitution of H18Y for T47I in CTB of El Tor strains was reported by Ansaruzzaman and colleagues [29]. Kumar et al. [30] reported a novel mutation from India at position 20 of CTB sequence (H20N) of the Vibrio cholerae O1 El Tor biotype [30].
In the present study, rCTB was expressed in E. coli M15 (pREP4) using pQE-30 vector. The sequence of ctxB gene was manipulated by a point mutation on 383 th nucleotide (guanine instead of cytosine) and consequently by serine-to-threonine substitution in mature CTB amino acids sequence at position 128. Moreover, the mutant retained pentameric stability and high affinity binding to anti-cholera toxin IgG used in Western-blot analysis. This nucleotide substitution was chosen because the subsequent substitution occurs between two amino acids which are in the same group with polar, uncharged R group in their chemical structure. Some properties of these two amino acids such as molecular weight and solubility are similar, therefore the expression of mutant rCTB without changing in conformation or biological properties could be guaranteed. It was illustrated that point mutation (serine-to-threonine substitution at position 128 on amino acid sequence of rCTB) caused overexpression of mutant rCTB (approximately 10 fold higher than mature rCTB). In addition, the expressed mutant rCTB retained pentameric stability and affinity to bind to anti-cholera toxin IgG. The over-expression of mutant rCTB in the present study may be probably due to the increase in the solubility of mutant protein via serine-to-threonine substitution and decrease in the amount of protein expressed as inclusion bodies.
Some different studies have investigated the effects of different mutations or manipulations of the CTB sequence. Merrite and colleagues [31] reported a point mutation at amino acid Gly(33) of the CTB sequence (Gly33Arg), which led to increase in the affinity of CTB to bind to receptors. In the study by Silva et al. [32], the substitution of G for A residue was performed by site-directed mutagenesis at 182 th nucleotide of native CTB sequence, which ultimately resulted in the production of glutamate instead of glycine. This process abolished the biding of CTB to GM1 without affecting the expression level of CTB protein.
Arêas and colleagues [19] were designed a native ctxB protein sequence according to the codon usage of E. coli, Lactobacillus casei, and Salmonella typhimurium. They manipulated the ctxB coding sequence by substitution of some nucleotides for the purpose of an over-expression of CTB protein in pAE-ctxB system [19].
Aman and colleagues [33] reported the engineering and crystallographic structure of a mutant cholera toxin, with histidine-to-alanine substitution at position 57 in the B subunit. Although the mutant protein retained its pentameric stability and high affinity binding to GM1 ganglioside, its immunomodulatory activity was lost due to this substitution [33].
Our previously published data have indicated the pQE-ctxB construct as a highly efficient system to produce rCTB [34] and high affinity binding to GM1 ganglioside [35]. Some advantages of this construct are as follow: i) pQE30 contains the T5 promoter that is recognized by the E. coli host RNA polymerase. This promoter is under the control of lac operon, which can be induced by IPTG and is one of the powerful promoters to express target genes. ii) The recombinant proteins, which were expressed with extra 6xHis-tag sequence at C-terminus of proteins in pQE vector, enable us to use Ni +2 -charged column chromatography to purify recombinant proteins. iii) The extra thrombin sequence, which was added to C-terminus of rCTB, can facilitate the separation of His-tag sequence from expressed protein.
In conclusion, pQE plasmid can express a high level of rCTB in E. coli M15 (pREP4) when S128T amino acid substitution was performed by PCR mutagenesis. E. coli still remains one of the most useful organisms to produce recombinant proteins compared to other bacteria, and this expression system can be used to produce rCTB in high yield with particular manipulations.